This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-101: Structural analysis of macromolecular complexes. We plan to develop a new method for atomic structure determination of proteins from electron scattering of oriented single molecules, thereby eliminating the reliance of crystallography on single crystals. In this project, protein ions generated from an electrospray ionization source will be embedded in a pulsed stream of superfluid helium droplets. The droplet beam containing the ions is to orthogonally intercept an elliptically polarized laser beam and a coherent high energy electron beam. The laser beam is to control the orientation of the ions. Oversampling of the continuous electron diffraction patterns under different orientations of the ions offers sufficient information for atomic structural determination. The extreme low temperature of the superfluid helium droplet beam, down to 0.38 K, is extremely beneficial for effective laser induced orientation and high resolution electron diffraction. Our plan is to demonstrate the feasibility of this idea within two years. During the first year, we will achieve orientation of native or near native protein ions embedded in superfluid helium droplets. We will first follow a documented design of the protein ion source to produce a high flux of native or near native protein ions. After the addition of a superfluid helium droplet source to intercept the protein ions for effective cooling, we will then add a detection chamber downstream from the droplet/ion intercept region, and use a laser to induce fluorescence from a few fluorescing proteins. This step is to confirm the conformation of the embedded ion. After introducing an orientation laser into the detection chamber, we can use linear dichroism spectroscopy of the fluorescing protein to measure the degree of orientation. In the meantime, we will modify an existing transmission electron microscope for pulsed electron diffraction. We can then assemble the complete experimental apparatus and use a few standard proteins for the experiment of "proof of concept". This step also involves using the phase retrieval and structure refinement software to obtain the atomic structure of the protein and compare the result with the available information from the protein databank. Several key technological developments over the past decade contribute to the timely success of this project. First of all, the PI's (Kong's) research group is the only group in the world specializing in field induced orientation and superfluid helium droplet cooling, from theoretical modeling to experimental observation. Secondly, the theoretical principle and experimental demonstration of phase retrieval from oversampling of continuous diffraction patterns have inspired the field of single molecule diffraction, with major investments in ultrafast x-ray free electron laser facilities throughout the world. Thirdly, the mass spectrometry community has taken great strides in generating near native proteins for secondary and higher order structure studies. By combining these technological breakthroughs from different fields, we hope to succeed in the ultimate conquest of crystallography without crystals. This project has the potential for shifting the paradigm of crystallography. Although the radiation source at this stage is pulsed coherent electron beams, the fundamental principle of operation is equally applicable to x-ray sources, either ultrafast or continuous. Ultimately, user facilities of this type can be established at beamlines such as the Stanford Linear Accelerator Center or National Synchrotron Light Source II. By matching the duty cycle of an electrospray ionization source with that of the radiation source, sample consumption can be reduced to femtomoles, a target achievable even for the most difficult protein to express. With further development in the spraying technology for macromolecules, protein complexes, and nanomaterials, the difficult and yet unpredictable process of crystal growth will no longer be mandatory. With tremendous savings in human effort, time, and money, daring hypotheses on disease mechanisms and radical therapeutic strategies could be tested from structural information in a timely and cost-effective manner. An idea would no longer have to be dismissed simply because one cannot justify a significant investment of time and effort needed to grow a sufficiently large sized single crystal for structural evidence. Decreasing or eliminating the size limit for crystallography can thus bring a fundamental transformation in the mindset of biological scientists. PUBLIC HEALTH RELEVANCE: If successful, this project has the potential to dramatically accelerate the rate of mechanistic and therapeutic studies for a gamut of diseases, including cancer, AIDS and diabetes. Most drug targets in pharmaceutical research involve proteins that are difficult or impossible to crystallize, and this project will eliminate the reliance of crystallography on single crystals. With tremendous savings in human effort, time, and money, daring hypotheses on disease mechanisms and radical therapeutic strategies could be tested from structural information in a timely and cost-effective manner.